Our laboratory has provided a unique perspective of endothelial health, showing that argininosuccinate synthase (AS) of the citrulline-nitric oxide cycle must be functional to sustain nitric oxide (NO) production and to maintain cell viability. Since virtually all phenotypic properties of normally functioning endothelial cells are related to the bioactivity of NO, impairment of NO production is a common mechanism by which cardiovascular risk factors, such as diabetes, mediate deleterious effects on the vascular wall. Based on our evidence that insulin regulates AS expression and serine-threonine phosphorylation, the first aim of this proposal is designed to explore the mechanisms by which insulin regulates vascular health and NO production through AS-dependent mechanisms. We will also explore the mechanisms that impair insulin function in disease by examining alterations to insulin regulation of AS under pathogenic conditions mediated by TNFa. Methodology used to investigate this specific aim will include: 1) post-translational modification analyses;2) signal transduction inhibitors in conjunction with siRNA;3) functional studies via enzyme activity and NO assays;4) fluorescence microscopy;5) real time PCR and western blotting;and 6) promoter analysis using mutagenesis, luciferase assays and EMSA studies. The second aim is based on our work demonstrating that endothelium-specific upstream open reading frame of AS mRNA encodes a small protein, Argininosuccinate Synthase Regulatory Protein (ARP), which regulates AS mRNA translation. We hypothesize that ARP represses AS expression, perhaps in response to pathogenic stimuli, by stalling AS mRNA particles, routing them into discrete cytoplasmic foci known as stress granules. To test this hypothesis, we will employ: 1) in vitro translation;2) RNA immunoprecipitation;3) RNA EMSA;4) polysome profiling analyses;5) stress granule formation and inhibition assays;and 6) immunofluorescence and in situ fluorescent staining. In the third specific aim, we will examine the molecular mechanisms underlying cardiovascular actions of insulin on AS expression and activity, the reciprocal relationships between insulin resistance and AS function, and implications for developing beneficial therapeutic strategies that simultaneously target metabolic and cardiovascular diseases using the rat diabetic model system. We will utilize two animal models of diabetes that are known to demonstrate NO-dependent endothelial dysfunction that will permit the process of translating the mechanisms defined in our tissue culture and in vitro systems into an whole animal system. Experimental approacheswillinclude:1) animal model characterization;2)real time PCR;3) westernblot;4) NO measurements and AS activity assay;5) immunofluorescence and in situ fluorescent staining. Overall, the work proposed is anticipated to provide an important and novel understanding of specific mechanisms that control vascular endothelial health. Results from this study have the potential to distinguish new therapeutic targets to be used to prevent or inhibit vascular endothelial dysfunction found in diabetes, obesity and other related vascular diseases. PUBLIC HEALTH RELEVANCE Impairment of vascular wall function has been suggested to be an early, causative event in diabetes, leading to atherosclerosis. Therefore, deciphering the cellular mechanisms that maintain vascular function is vital to understanding how physiologic affecters mediate their protective effects or their deleterious effects on the vascular wall. We believe examination of the function and regulation of a critical enzyme that is required to maintain vascular wall function is essential to this understanding, and will potentially distinguish new therapeutic strategies for the treatment of diabetes, hypertension and related cardiovascular disease